RhoG facilitates a conformational transition in the guanine nucleotide exchange factor complex DOCK5/ELMO1 to an open state

The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac. RhoG, another GTPase, activates the ELMO-DOCK-Rac pathway during engulfment and migration. Recent cryo-EM structures of the DOCK2/ELMO1 and DOCK2/ELMO1/Rac1 complexes have identified closed and open conformations that are key to understanding the autoinhibition mechanism. Nevertheless, the structural details of RhoG-mediated activation of the DOCK/ELMO complex remain elusive. Herein, we present cryo-EM structures of DOCK5/ELMO1 alone and in complex with RhoG and Rac1. The DOCK5/ELMO1 structure exhibits a closed conformation similar to that of DOCK2/ELMO1, suggesting a shared regulatory mechanism of the autoinhibitory state across DOCK-A/B subfamilies (DOCK1−5). Conversely, the RhoG/DOCK5/ELMO1/Rac1 complex adopts an open conformation that differs from that of the DOCK2/ELMO1/Rac1 complex, with RhoG binding to both ELMO1 and DOCK5. The alignment of the DOCK5 phosphatidylinositol (3,4,5)-trisphosphate binding site with the RhoG C-terminal lipidation site suggests simultaneous binding of RhoG and DOCK5/ELMO1 to the plasma membrane. Structural comparison of the apo and RhoG-bound states revealed that RhoG facilitates a closed-to-open state conformational change of DOCK5/ELMO1. Biochemical and surface plasmon resonance (SPR) assays confirm that RhoG enhances the Rac GEF activity of DOCK5/ELMO1 and increases its binding affinity for Rac1. Further analysis of structural variability underscored the conformational flexibility of the DOCK5/ELMO1/Rac1 complex core, potentially facilitating the proximity of the DOCK5 GEF domain to the plasma membrane. These findings elucidate the structural mechanism underlying the RhoG-induced allosteric activation and membrane binding of the DOCK/ELMO complex.

The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac.RhoG, another GTPase, activates the ELMO-DOCK-Rac pathway during engulfment and migration.Recent cryo-EM structures of the DOCK2/ELMO1 and DOCK2/ELMO1/Rac1 complexes have identified closed and open conformations that are key to understanding the autoinhibition mechanism.Nevertheless, the structural details of RhoG-mediated activation of the DOCK/ELMO complex remain elusive.Herein, we present cryo-EM structures of DOCK5/ ELMO1 alone and in complex with RhoG and Rac1.The DOCK5/ ELMO1 structure exhibits a closed conformation similar to that of DOCK2/ELMO1, suggesting a shared regulatory mechanism of the autoinhibitory state across DOCK-A/B subfamilies (DOCK1−5).Conversely, the RhoG/DOCK5/ELMO1/Rac1 complex adopts an open conformation that differs from that of the DOCK2/ELMO1/Rac1 complex, with RhoG binding to both ELMO1 and DOCK5.The alignment of the DOCK5 phosphatidylinositol (3,4,5)-trisphosphate binding site with the RhoG Cterminal lipidation site suggests simultaneous binding of RhoG and DOCK5/ELMO1 to the plasma membrane.Structural comparison of the apo and RhoG-bound states revealed that RhoG facilitates a closed-to-open state conformational change of DOCK5/ELMO1.Biochemical and surface plasmon resonance (SPR) assays confirm that RhoG enhances the Rac GEF activity of DOCK5/ELMO1 and increases its binding affinity for Rac1.Further analysis of structural variability underscored the conformational flexibility of the DOCK5/ELMO1/Rac1 complex core, potentially facilitating the proximity of the DOCK5 GEF domain to the plasma membrane.These findings elucidate the structural mechanism underlying the RhoG-induced allosteric activation and membrane binding of the DOCK/ELMO complex.
Rho GTPases are key regulators of cytoskeletal dynamics that affect many cellular processes, such as morphogenesis, polarity, motility, and cell division (1).Dedicator of cytokinesis (DOCK) proteins are evolutionarily conserved guanine nucleotide exchange factors (GEFs) for Rho GTPases Rac and Cdc42, facilitating the conversion of these GTPases from GDP-to GTP-bound forms (2,3).Eleven mammalian DOCK proteins regulate various normal and pathological processes of development and immunity by activating Rac and Cdc42 (4,5).
Recent cryo-EM structures of the DOCK2/ELMO1 complex in the Rac1-bound and unbound states revealed that autoinhibitory regulation occurs through conformational changes in the ELMO1 NTD (21).In addition, the crystal structures of the RhoG/ELMO RBD and BAI1/ELMO2 NTD complexes were determined (21)(22)(23).Based on these structures, it was hypothesized that the binding of RhoG and/or BAI1 to ELMO1 NTD would release DOCK2/ELMO1 from autoinhibition and promote its localization to the plasma membrane.However, the structure of the DOCK/ELMO complex bound to RhoG and other upstream proteins has not yet been elucidated.
Our previous study illustrated the cryo-EM structure of the DOCK5/ELMO1/Rac1 complex (24), which is a more curved dimer than the DOCK2/ELMO1/Rac1 complex.However, the significance of the observed structural difference remains unclear.ELMO1 NTD appeared disordered in the DOCK5/ ELMO1/Rac1 complex, whereas in the DOCK2/ELMO1/ Rac1 complex, it was structured into an S-shaped open conformation (21).In the S-shaped conformation of ELMO1 NTD , the symmetric DOCK2/ELMO1 dimer and RhoG cannot simultaneously interact with the membrane (25).Therefore, it remains unclear how the DOCK/ELMO complex is activated by membrane-localized RhoG.To address these questions, we determined the cryo-EM structures of the DOCK5/ELMO1 complex alone and bound to RhoG and Rac1.

Results and discussion
The structure of DOCK5/ELMO1 We initially determined the cryo-EM structure of the DOCK5/ELMO1 binary complex using human full-length DOCK5 (DOCK5 FL , residues 1−1870) and ELMO1 (ELMO1 FL , residues 1−727) proteins (Fig. 1A).Our first attempt to analyze the DOCK5 FL /ELMO1 FL complex produced a low-resolution (7.3 Å) cryo-EM map with two-fold symmetry but lacked density for ELMO1 NTD (Fig. S1).We next analyzed a DOCK5 FL /ELMO1 FL sample crosslinked with bis(sulfosuccinimidyl)suberate (BS 3 ), a widely used Nhydroxysuccinimide ester-based crosslinking agent (26), achieving a reconstruction at improved resolution (5.8 Å) that clearly showed the ELMO1 NTD density with two-fold symmetry (Figs.S1 and S2).To enhance local resolution, we symmetrically expanded and refined the particles, focusing on a single DOCK5/ELMO1 protomer and the dimerization component, which led to structure determination at 4.8 Å resolution (Fig. S2 and Table S1).Model building and refinement of the DOCK5 FL /ELMO1 FL complex were completed, with the exception of the C-terminal region of DOCK5 (residues 1643−1870), which remained disordered.
The structure of DOCK5/ELMO1 showed a closed conformation similar to that reported for DOCK2/ELMO1 (21) (Fig. 1B).The a-solenoid of ELMO1 NTD was clamped onto the DHR-2 domain of DOCK5, occluding the Rac1 binding site.This indicates that the DOCK1−5/ELMO1 complexes likely have autoinhibitory regulation in common.
Despite the common closed conformation, a detailed structural comparison revealed that DOCK5/ELMO1 is more compact than DOCK2/ELMO1, with the two ELMO1 NTDs located closer to each other (Fig. 2A).A closer interaction was observed for DOCK5 than for DOCK2 at the ELMO1 RBD -DHR-2 interface, between Glu36 and Asp39 in ELMO1 and His1545 in DOCK5 (Fig. 2B).The observed differences are unlikely to be due to the crosslinking of DOCK5/ELMO1, as crosslinked samples were similarly used in the cryo-EM analysis of DOCK2/ELMO1 (21).We performed structure-based mutagenesis at the ELMO1 RBD -DOCK5 DHR-2 interface to examine the GEF activity of DOCK5 FL /ELMO1 FL against Rac1 (GTPase domain, residues 1−177).Simultaneous mutation of Glu36 and Asp39 in ELMO1 to alanine (DOCK5/ELMO1 E36A/ D39A ) resulted in a 1.5-fold increase in GEF activity for Rac1 compared to DOCK5/ELMO1 WT (Fig. 2C).This suggests that these ELMO1 residues are involved in the DOCK5/ELMO1 autoinhibitory interaction.His1545 in DOCK5 is not conserved in the DOCK-A/B subfamilies from DOCK1 to DOCK4 (Fig. 2D), suggesting that the autoinhibitory conformation may be specifically stabilized in DOCK5.

RhoG activates DOCK5/ELMO1 by promoting its binding to Rac1
Next, we examined the GEF activity of DOCK5 FL /ELMO1 FL against Rac1 in the presence of the constitutively active RhoG Q61L mutant (GTPase domain, residues 1−184).The GEF activity of DOCK5 FL /ELMO1 FL increased with increasing RhoG Q61L concentration, reaching half-maximum at 4.6 mM (Fig. 3, A and B).This value is in close agreement with the binding affinity (K D = 10 mM) of RhoG Q61L for DOCK5 DC / ELMO1 FL , a complex of C-terminal truncated DOCK5 (DOCK5 DC , residues 1−1642) and full-length ELMO1 (Fig. 1A), as determined by the surface plasmon resonance (SPR) (24), indicating that DOCK5/ELMO1 is activated by binding to the active form of RhoG, as expected.
We also used SPR to examine the effect of RhoG Q61L on the Rac1 binding affinity for DOCK5 FL /ELMO1 FL .We immobilized Rac1 (residues 1−188) on an SPR sensor chip and passed varying concentrations of DOCK5 FL /ELMO1 FL in the absence or presence of 10 mM RhoG Q61L to measure the rates of association and dissociation (Fig. 3C).DOCK5 FL /ELMO1 FL showed a higher affinity for Rac1 in the presence of RhoG Q61L (K D = 0.034 nM) than in its absence (K D = 0.15 nM) (Fig. 3D).Collectively, these results suggest that RhoG activates DOCK5/ ELMO1 by facilitating its binding to Rac1.
The cross-linked RhoG Q61L /DOCK5 DC /ELMO1 FL /Rac1 G15A sample yielded a two-fold symmetrical reconstruction at a resolution of 3.8 Å (Fig. S3).However, the ELMO1 NTD was poorly resolved due to its high mobility.To address this, we symmetrically expanded and refined the particles focusing on ELMO1 NTD (Fig. S3).Through 3D classification, excluding the DOCK5 DHR-2/Rac1 segment, ELMO1 NTD was resolved into several conformations.One class, comprising approximately 12% of the particles, provided a map of sufficient quality to define the structure of DOCK5/ELMO1 bound to RhoG at 4.9 Å resolution (Fig. S3 and Table S1).Cross-linking mass spectrometry confirmed RhoG's placement within the cryo-EM map (Fig. S4 and Table S2).From this partial map, we reconstructed the entire RhoG/DOCK5/ELMO1/Rac1 complex at 4.7 Å resolution with an ordered RhoG-ELMO1 NTD (Fig. S5 and Table S1).Further reconstruction with both ordered RhoG-ELMO1 NTD yielded similar maps at resolutions of 8.2 Å and 7.3 Å with C1 and C2 symmetry, respectively (Fig. S5).
In the RhoG-bound DOCK5 DC /ELMO1 FL /Rac1 G15A complex, a curved dimer formed, with RhoG located at both protomer tips (Fig. 1C).Rac1 interacted with the DHR-2 domain of DOCK5 and the PH domain of ELMO1 near the dimer interface, akin to the prior DOCK5 DC /ELMO1 FL /Rac1 G15A ternary complex (24).Notably, ELMO1 NTD was disordered in the previous ternary structure.However, in the present RhoGbound structure, the a-solenoid of ELMO1 NTD arched toward the DHR-1 domain of DOCK5 and bound to RhoG in cooperation with the C2 domain of DOCK5 (Fig. 1C).Due to this binding mode, the ELMO1 NTD of the RhoG/DOCK5/ELMO1/ Rac1 complex had a compact, open conformation that was different from the elongated (S-shaped) open conformation of the DOCK2/ELMO1/Rac1 complex (Fig. S6).In the present RhoG/DOCK5/ELMO1/Rac1 structure, the C-terminus of RhoG, Pro181, near the lipidation site, is adjacent to the phosphatidylinositol (3,4,5)-trisphosphate binding site of DOCK5, which includes basic residues such as Lys457, Lys460, and Lys464 (Fig. 4A).Four membrane-binding sites (two DHR-1 and two RhoG) were aligned in the same direction in the dimeric configuration.Thus, the present structure suggests that RhoG and the symmetric DOCK5/ELMO1 dimer can simultaneously bind to the plasma membrane.
the GDP-bound RhoG (24).By contrast, the secondary interface consisted of aromatic or aliphatic amino acid residues, such as Val232, Phe345, and Trp400 of DOCK5, which interacted with Arg127 and Lys130 of the Rho insertion region (a3 0 helix) of RhoG Q61L (Fig. 4C).Thus, the secondary interface appears to be independent of the nucleotide-binding state of RhoG.The C2 domain of DOCK5 interacted with the ELMO domain of ELMO1 in the apo state of DOCK5/ELMO1 (Fig. 4, D and E).Therefore, DOCK5 C2 may have broad specificity in protein-protein interactions.
We performed biochemical experiments to evaluate the RhoG binding interface observed in the cryo-EM structure.Because the RhoG-ELMO1 RBD interaction is constituted by the RhoG switch regions that undergo nucleotide-dependent conformational changes (Fig. 4B), we first assessed the ability of GDP-bound or GTPgS-bound RhoG WT to activate DOCK5 FL /ELMO1 FL in an in vitro GEF assay (Fig. 4F).GTPgS-bound RhoG enhanced the GEF activity against Rac1 in DOCK5 FL /ELMO1 FL by 2.06-fold, whereas GDP-bound RhoG enhanced it only slightly (by 1.15-fold) as expected.This result confirmed the importance of the RhoG-ELMO1 RBD interaction in the activation of DOCK5/ELMO1 by RhoG.
Next, we assessed the RhoG-DOCK5 C2 interaction by structure-based mutagenesis: Arg127 and Lys130 of RhoG G61L at the DOCK5 interface were simultaneously mutated to alanine (R127A/K130A) (Fig. 4C), and its ability to activate DOCK5 FL /ELMO1 FL was examined.The control RhoG Q61L enhanced the GEF activity of DOCK5 FL /ELMO1 FL by 2.07fold, whereas the R127A/K130A mutant of RhoG Q61L enhanced it by 1.67-fold, a 20% reduction compared to RhoG Q61L (Fig. 4G).These results indicate that the RhoG-DOCK5 C2 interaction contributes to the activation of DOCK5/ ELMO1 by RhoG in an in vitro system.

DOCK5/ELMO1/Rac1 flatter conformation
The RhoG-bound DOCK5/ELMO1/Rac1 dimer exhibited a similar curved structure as the unbound DOCK5/ELMO1/ Rac1, while the DOCK2/ELMO1/Rac1 dimer exhibited a flatter structure (Fig. 5A).We thus evaluated conformational heterogeneity as a possible cause of these structural differences.We performed 3D variability analysis using the 3.8 Åresolution consensus map of RhoG/DOCK5/ELMO1/Rac1.This analysis revealed several continuous movements within individual protomers of the DOCK5/ELMO1 CTD /Rac1 core region (Movies S2 and S3).The major movement was that DOCK5 showed flexibility between N-and C-terminal modules at the boundary between armadillo repeat domain and DHR-2.
Next, to capture representative conformations of the DOCK5/ELMO1 CTD /Rac1 core region, we divided the RhoG/ DOCK5/ELMO1/Rac1 consensus map into 10 classes by 3D classification (Fig. S7).Five classes of sufficient quality were obtained with resolutions ranging from 4.2 to 4.5 Å and subjected to atomic model building and refinement (Table S3).The resulting structures of classes 1 to 5 differed from one another in dimeric molecular dimensions (Fig. 5B).The distance between the two DHR-1 of DOCK5 at the tip of the DOCK5/ELMO1 CTD /Rac1 protomers, measured between the Ca atoms of Lys460 (one of the phosphatidylinositol (3,4,5)trisphosphate binding residues), was shortest for class 1 and longest for class 5. Structural differences were due to the conformational changes between the armadillo repeat domain and DHR-2 in DOCK5, accompanied by a shift in the PH domain of ELMO1 (Fig. 5C).Of the five, class 5 of DOCK5/ ELMO1 CTD /Rac1 showed a flatter dimer than that of the others, similar to the DOCK2/ELMO1/Rac1 dimer (Fig. 5, A  and B).Collectively, these results suggest that DOCK2/ ELMO1 and DOCK5/ELMO1 are intrinsically similar in structure and exhibit conformational flexibility.
Our previous study of the DOCK5/ELMO1/Rac1 ternary complex (without RhoG) focused on obtaining the highest resolution map for de novo model building and did not detect conformational flexibility.However, since RhoG binding has little effect on the overall conformation of the DOCK5/ ELMO1/Rac1 core region (Fig. 5A), it is possible that conformational changes similar to those in this study could occur even without RhoG binding.
In summary, the structural, biochemical, and biophysical analyses presented here reveal the RhoG-regulated allosteric mechanism of DOCK5/ELMO1 activation.Although this study is based on a simplified system using soluble RhoG and Rac1 proteins lacking C-terminal lipidation, the binding mode of RhoG suggests that DOCK5/ELMO1 activation occurs at the plasma membrane (Fig. 6).In contrast, the Rac1 binding site in DOCK5/ELMO1 is located in the concavity of the curved dimer, away from the predicted membrane surface.This suggests that the DHR-2 domain must be substantially rearranged during the membrane recruitment of DOCK5/ ELMO1 to enable binding to membrane-localized Rac1.Consistent with this theory, our cryo-EM single particle analysis reveals that the DOCK5/ELMO1 CTD /Rac1 core region is highly flexible and can, therefore, readily transition to a flatter conformation.We hypothesize that the flexibility observed in DOCK5 may allow the DHR-2 domain to approach the membrane-localized Rac under intracellular conditions.The lack of comprehensive knowledge of the conformational changes in the DOCK/ELMO complex on the membrane is attributed to limitations associated with current soluble protein-based experimental strategies.Structural analysis using lipid membranes and lipidated Rho GTPases will pave the way for understanding the molecular mechanisms underlying DOCK-mediated Rac activation in cells.

Cryo-EM sample preparation, data collection, and data processing
Three microliters of protein sample (diluted to 200−400 nM in 20 mM HEPES-NaOH pH 8.0, 300 mM NaCl, 1 mM TCEP, and 0.06% digitonin) were applied to the glow-discharged holey carbon grids (Quantifoil R1.2/1.3,Cu, 300 mesh) supported with a thin film of carbon or graphene.Grids were incubated for 30 s at 4 C and 100% humidity, blotted for 1 s, and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific).The data were collected on a 300 kV Titan Krios G4 electron microscope (Thermo Fisher Scientific) using a K3 direct electron detector (Gatan) in counting mode.Micrograph movies were acquired at a nominal magnification of 64,000×, corresponding to a calibrated pixel size of 1.33 Å per pixel.Each movie was recorded for 4.1 s and subdivided into 50 or 48 frames.The electron flux rate was set to 21.4 to 21.8 e − per pixel/second at the detector, resulting in an accumulated exposure of 50 e − /Å 2 at the specimen.The data were automatically acquired through the image shift method using EPU software with a defocus range of −0.8 to −2.0 mm.The collected movies were motion-corrected using MotionCor2 (29) with dose weighting.Parameters of the contrast transfer function were estimated from the motioncorrected micrographs using CTFFIND4 (30).Particles were automatically picked using crYOLO (31), and subsequent 2D and 3D analyses were performed using Relion 3.1 (32).Detailed data processing schemes for the DOCK5 FL /ELMO1 FL and RhoG Q61L /DOCK5 DC /ELMO1 FL /Rac1 G15A complexes are shown in Figs S2-S5, respectively.Moreover, 3D continuous conformational heterogeneity was evaluated by 3D variability analysis in cryoSPARC (33).

Model building and refinement
The atomic model was built using the structures of the DOCK5 DC /ELMO1 FL /Rac1 G15A complex (PDB ID: 7DPA) and the ELMO1 RBD -RhoG complex (PDB ID: 7Y4A).The Alpha-Fold2 model (34) was utilized to further improve DOCK5 sequence assignments, especially in the C2 domain.The ELMO1 region 82−530 was modeled based on sequence homology with the corresponding region of ELMO2 (Protein Data Bank (PDB) ID: 6IE1).The model was fitted to the cryo-EM density map as a rigid body using UCSF Chimera (35) and manually adjusted using COOT (36).The final model was refined using Phenix (37).

In vitro GEF assays
All measurements of the nucleotide exchange reaction were performed using the full-length DOCK5 FL /ELMO1 FL complex and the Rac1 GTPase domain (residues 1−177).For DOCK5/ ELMO1 WT or DOCK5/ELMO1 E36A/D39A GEF assays, each DOCK5/ELMO1 (25 nM) or control buffer was incubated with

SPR
SPR experiments were performed using a Biacore T200 instrument (Cytiva).Rac1 (residues 1-188) was immobilized on a Series S Sensor Chip CM5 using the Amine Coupling Kit (Cytiva).Five different concentrations (0.37, 1.1, 3.3, 10, and 30 nM) of the full-length DOCK5 FL /ELMO1 FL complex were continuously injected in the presence and absence of 10 mM RhoG Q61L .Next, the responses were measured in buffer containing 10 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM MgCl 2 , 100 mM GTP, and 0.005% surfactant P-20.Data were processed through single-cycle kinetic analysis using the manufacturer's software.The kinetic parameters were estimated from three independent experiments.

Statistical analyses
Data were presented as mean ± SD considering three independent experiments.Two samples were statistically compared using an unpaired two-sided Student's t test; statistical significance was considered at p < 0.05.

Figure 1 .
Figure 1.Structures of the DOCK5/ELMO1 complexes.A, a schematic diagram of the domain boundaries of human DOCK5, ELMO1, RhoG, and Rac1.Black bars indicate the protein constructs used in this study.Asterisks (DOCK5, blue; ELMO1, red) indicate the hinges of the conformational changes observed in this study.B and C, Cryo-EM density (composite) maps and atomic models of the DOCK5/ELMO1 (B) and RhoG/DOCK5/ELMO1/Rac1 (C) complexes in two orthogonal views.Asterisks indicate the location of the hinges shown in A. ARM, armadillo repeat domain; DHR: dock homology region; EID, Elmo inhibitory domain; ELMO, Elmo domain; PH, Pleckstrin homology domain; PxxP, proline-rich region; RBD, Ras-binding domain; SH3, Src homology domain 3.
and Movie S1).RhoG interacted with ELMO1 RBD on one side and with DOCK5 C2 on the other side to mediate an open conformation of DOCK5/ ELMO1.These structures suggest that RhoG facilitates the conformational change of DOCK5/ELMO1 from the closed to the open state, thereby relieving the Rac1-binding site in DOCK5 DHR-2 from autoinhibition, consistent with the results of biochemical and SPR binding analyses (Fig. 3, A−D).

Figure 3 .
Figure 3. Biochemical characterization and conformational changes of DOCK5/ELMO1.A, GEF activity of DOCK5/ELMO1 against Rac1 with the addition of indicated concentrations of RhoG Q61L .B, effects of RhoG Q61L concentration on the biochemical activity of DOCK5/ELMO1 analyzed from the experiments shown in A. The activity was normalized relative to that at 0 mM RhoG Q61L .Data are presented as mean ± SD (n = 3 independent experiments).C, binding of DOCK5/ELMO1 to Rac1-immobilized SPR biosensor in the absence (left) and presence of RhoG Q61L at 10 mM (right).D, kinetic parameters of DOCK5/ELMO1 binding to Rac1 estimated from the experiments shown in C. k on , association rate constant; k off , dissociation rate constant; K D = k off /k on , equilibrium dissociation constant.Data are presented as mean ± SD (n = 3 independent experiments).E, a comparison of the protomer structures of DOCK5/ELMO1 (left) and RhoG/DOCK5/ELMO1/Rac1 (right).DOCK5 is depicted in cyan, ELMO1 in orange except for the red RBD, Rac1 in yellow, and RhoG in green.

Figure 4 .
Figure 4. RhoG interaction sites in DOCK5/ELMO1.A, structure of the RhoG/DOCK5/ELMO1/Rac1 complex from two orthogonal views.The locations of membrane-binding sites (the DOCK5 PIP3-binding residue Lys460 and the RhoG C-terminus Pro181) are indicated by black and green circles, respectively.B, a close-up view of the RhoG-ELMO1 RBD interface boxed in A. RhoG is shown in green except for switch 1 (magenta) and switch 2 (blue).C, a close-up view of the DOCK5 C2 -RhoG interface boxed in A. D, structure of the DOCK5/ELMO1 complex.The molecular orientation of DOCK5 is the same as the right panel in A.

Figure 5 .
Figure 5. Structural plasticity in DOCK5.A, comparison of the RhoG/DOCK5/ELMO1/Rac1 complex (this study, left) with the DOCK5/ELMO1/Rac1 complex (PDB ID: 7DPA) and the DOCK2/ELMO1/Rac1 complex (PDB ID: 6TGC, ELMO1 NTD omitted for clarity).B, comparison of five classes of the DOCK5/ELMO1 CTD / Rac1 core structures.The location of the Lys460 residue of DOCK5 in each protomer is indicated by a black circle.C, structural superimposition of two of five classes (class 1 and class 5 with the same color scheme as in B, class 1 is shown as transparent) in two orthogonal views.Blue asterisks indicate the location of the hinge between ARM and DHR-2 in DOCK5.

Figure 6 .
Figure 6.A proposed structural model of RhoG-mediated DOCK5/ELMO1 activation.A, the DOCK5/ELMO1 dimer localizes to the cytoplasm in the resting state.A subpopulation of DOCK5/ELMO1 is in a closed conformation in which the Rac1-binding site is occluded by the ELMO1 RBD.B, when RhoG is activated, the GTP-bound form of RhoG binds to ELMO1 RBD, shifting DOCK5/ELMO1 into an open conformation with the Rac1 binding site exposed.C, a hypothetical model of the DOCK5/ELMO1 CTD core approaching Rac1.A conformational change at the DOCK5 hinge (between ARM and DHR-2, blue asterisks) would bring DHR-2 closest to membrane-localized Rac1 for the GEF reaction.In each panel, the protomers on the right are shown as transparent.